A Faster, Simpler Way to Analyze DNA Replication
Simplifying Cell Proliferation Analysis
Compared to tedious BrdU assays, new, simplified Click-iT EdU flow cytometry kits make it even easier to detect and quantify DNA synthesis. The new assay kits contain 50% fewer components and a streamlined experimental protocol to make direct S-phase DNA measurement in proliferating cells simpler than ever.
Evaluate Cell Proliferation
The proportion of cells in the DNA synthesis phase provides a reliable indication of the proliferative capacity of a cell population. Click-iT EdU can be used to evaluate proliferation dynamics in primary human cells (an important experimental model). As shown in Figure 1, for Gibco Human Skeletal Myoblasts (HSkM) plated at three different densities and grown in differentiation media for 48 hours, higher plating densities correlated with a lower proliferative fraction of cells.
Figure 1. Correlation between HSkM plating density and proliferative fraction. Gibco Human Skeletal Myoblasts (HSkM) were thawed and plated in low-glucose DMEM plus 2% horse serum at (A) 10,000 cells/cm2, (B) 25,000 cells/cm 2 or (C) 50,000 cells/cm2. These plots show a correlation between cell plating density and proliferative fraction, with the percentage of EdU-positive cells decreasing as plating density increases. Cells were grown for 48 hr at 37°C/5% CO2, then fed a 10 μM pulse of EdU 2 hr prior to analysis. EdU incorporation into newly synthesized DNA was detected using a click reaction with Alexa Fluor 488 dye azide. Cells were analyzed with the Attune Acoustic Focusing Cytometer with a 488 nm laser using the standard BL1 (530/30) emission filter.
Detect Variation in Proliferative Cells
Click-iT EdU can also be used to detect variations in the proliferative fraction of cells due to cell cycle disruption. Figure 2 shows how Click-iT EdU assays were used with several cultured cell lines to directly measure the percentage of cells in S phase. Dual-parameter analysis of DNA content vs. Click-iT EdU fluorescence clearly identifies cells in different phases of the cell cycle. The percentage of cells in S phase provides an indication of the degree of cell proliferation.
Figure 2. Dual-parameter analysis of cell cycle perturbation. Gibco Primary Adult Human Dermal Fibroblasts (HDFa), cervical carcinoma cells (HeLa), and human alveolar epithelial cells (A549) were grown in culture for 48 hr. Cells were either treated with a 500 nM pulse of the antimitotic drug Paclitaxel or left untreated. Cells were treated with 10 µM EdU for 2 hr prior to analysis. Incorporated EdU was detected using a reaction with Click-iT EdU Alexa Fluor 488 dye azide. Cells were analyzed using the Attune Acoustic Focusing Cytometer with 405 nm and 488 nm lasers using standard VL1 (450/40) and BL1 (530/30) emission filters. Dual-parameter density plots of FxCycle™ Violet vs. Alexa Fluor 488 Click-iT EdU fluorescence clearly identified cells in the G0/G1, S, and G2/M phases of the cell cycle. Untreated A549 cells had a significantly higher percentage of cells in S phase and a lower percentage in G0/G1 than other cell types, reflecting a higher proliferative index. Upon treatment with paclitaxel, more than 90% of A549 and HeLa cells were arrested in G2 phase due to mitotic inhibition. A similar pattern of arrest was seen with HDFa, with the exception of approximately 26% of cells which remained in the quiescent G0 state.
An Easier Way to Analyze Cell Proliferation
The unique chemistry of assays based on Click-iT EdU technology enables a simple and efficient method for evaluating cell proliferation. These new assays help researchers to accurately correlate proliferative dynamics with different plating densities, and clearly identify cells in the S phase of the cell cycle. Click-iT EdU labeling is compatible with most fixation protocols.
For Research Use Only. Not intended for any animal or human therapeutic or diagnostic use.
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